GSM6893668: GSC_CW468_shRNA_shSNX10.358_Replicate_1; Homo sapiens; RNA-Seq
For SNX10 knockdown experiments, Trizol reagent (Sigma Aldrich) was used to isolate total cellular RNA from cell pellets according to the manufacturer's instructions. Three patient-derived GSCs were utilized with a non-targeting shRNA control and three independent non-overlapping shRNAs targeting SNX10 for each GSC. Biologic duplicates were utilized for each GSC. RNA was purified using an mRNAeasy kit (Qiagen) and subjected to RNA-sequencing by Novogene Corporation. After the QC procedures, mRNA from eukaryotic organisms is enriched using oligo(dT) beads. For prokaryotic organisms or eukaryotic organisms' long-non-coding libraries, rRNA is removed using the Ribo-Zero kit that leaves the mRNA. First, the mRNA is fragmented randomly by adding fragmentation buffer, then the cDNA is synthesized by using mRNA template and random hexamers primer, after which a custom second-strand synthesis buffer (Illumina) , dNTPs, RNase H and DNA polymerase I are added to initiate the second-strand synthesis. Second, after a series of terminal repair, A ligation and sequencing adaptor ligation, the double-stranded cDNA library is completed through size selection and PCR enrichment.